Monoclonal antibody against human IgE

ABSTRACT

The present invention provides novel monoclonal antibodies HE-22A, HE-35A, HE-39E, and HE-69B against human IgE, a mixture thereof, hybridomas producing the antibodies, and immunoassays of human IgE employing the antibodies, which are useful for clinical diagnosis of allergic diseases or parasitic infections.

This application is a continuation, of application Ser. No. 07/686,405filed on Apr. 17, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies against humanimmunoglobulin E (IgE), a mixture thereof, hybridomas producing themonoclonal antibodies, and immunoassays of human IgE employing themonoclonal antibodies. The monoclonal antibodies according to thepresent invention are useful for clinical diagnostic agents to determinehuman IgE.

Prior Art

IgE is an immunoglobulin identified in 1966 by Ishizaka et al and itbinds to mast cell or basophil surface via Fc receptor and reacts withallergens, thereby causing an immediate-type allergy. An IgE level inblood is quite low as compared with other immunoglobulins, but itsignificantly increases in the cases of allergic diseases, hepaticfailure and parasitic infections, thus being characterized by the broadrange of the levels in blood. Accordingly, to determine IgE level inblood is useful for diagnosis of allergic diseases or parasiticinfections. The methods commonly used for this purpose includeradioimmunoassay (RIA) and enzyme immunoassay (EIA) methods.

Monoclonal antibodies specifically reacting with human IgE have alreadybeen reported by Ichimori et al (Hybridoma, 4, 47 (1985)) and a methodfor determining human IgE in blood employing human IgE-specificmonoclonal antibodies has also been reported by J. Grassi et al (J.Allergy. Clin. Immunol., 77, 808 (1986)). However, when such aconventional method employing monoclonal antibodies is used to determinehuman blood IgE, problems such as limited range of the determination andcomplicated determination procedure are experienced. Further, since thecalibration curve is not always linear, the sensitivity is varieddepending on the range of the concentrations to be determined and allthe concentrations so determined are not reliable. The conventionalmethods also require special devices. All these problems andinconvenience can not be eliminated at a stretch by the conventionalmethods.

SUMMARY

Human IgE-specific monoclonal antibodies HE-22A, HE-35A, HE-39E, andHE-69B provided by the present invention are characterized in that anyone of the four monoclonal antibodies binds to human IgE at the regiondifferent from those the other three bind to without the one receivingany interference from the other three antibodies. Further, the 4monoclonal antibodies bind to human IgE with different affinities sothat the reactivity of a mixture of the antibodies with the human IgEcan be controlled by appropriately mixing them. Based upon theseproperties, the human IgE-specific antibodies of the present inventioncan be used to determine quite easily human IgE levels of a wide rangein blood with higher reliability without using any special devices.Accordingly, the monoclonal antibodies of the present invention canroutinely be used as clinical diagnostic agents to determine human IgE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactivity of each monoclonal antibody according to thepresent invention with heat-treated IgE.

FIG. 2 shows sensitivity curves for IgE in sandwich ELISA system usingcombinations of the present monoclonal antibodies.

FIG. 3 shows the sensitivity curves of each of 3 monoclonal antibodies(HE-22A, HE-35A and HE-39E) and of the mixture of these three antibodiesin sandwich RIA system using the monoclonal antibody HE-69B-immobilizedbeads.

DETAILED DESCRIPTION OF THE INVENTION

Human IgE-specific monoclonal antibodies provided by the presentinvention are characterized in that any one of the four monoclonalantibodies binds to human IgE at the region different from those theother three bind to without the one receiving any interference from theother three of the antibodies. In addition, the 4 human IgE-specificmonoclonal antibodies bind to human IgE with different! affinities sothat the reactivity of a mixture of the antibodies to the human IgE canbe controlled by appropriately mixing them. Based on these properties,human IgE-specific antibodies according to the present invention can beused to determine quite easily a wide range of human IgE levels in bloodwith a higher reliability without using any special devices.Accordingly, the monoclonal antibodies of the present invention canroutinely be used as clinical diagnostic agents to determine human IgE.

1) Preparation of hybridoma

Mice are immunized with human IgE. Cells capable of producing antibodiesare obtained from the immunized mice and fused with myeloma cells, andresulting hybridoma cells are screened for the production of antibodieswhich react highly with the immunogen human IgE and differently derivedhuman IgE but not with human IgG. Hybridomas selected are cloned andexamined intensively for the reaction specificity of the producedantibody with 3 differently derived human IgEs, human IgA, human IgM,and human IgG, and clones producing antibodies which react highly withthe immunogen human IgE and differently derived human IgEs but not withother human immunoglobulins are selected and incubated to yieldmonoclonal antibodies. The procedures of immunization, cell fusion, andscreening of fused cells can be conducted by standard methods.

2) Preparation of monoclonal antibodies

Selected hybridomas are proliferated by in vitro or in vivo incubationto obtain monoclonal antibodies. In the case of in vitro incubation,hybridomas are incubated in complete RPMI medium until the proliferationlimit is achieved and then the supernatant of the medium is recovered,from which monoclonal antibodies are obtained. In the case of in vivoincubation, BALB/c mice previously administered with pristaneintraperitoneally are transplanted with 5×10⁶ -10⁷ hybridoma cellsintraperitoneally and about 3 weeks after the transplantation the miceare observed to ensure the swelling of the abdomens and ascitic fluid istaken, from which IgG fraction is isolated to obtain monoclonalantibodies.

In the present invention, according to the method mentioned above wereobtained the monoclonal antibodies HE-39E, HE-22A, HE-69B, and HE-35Awhich specifically bind to human IgE. Hybridomas HE-39E Shionogi, HE-22AShionogi, HE-69B Shionogi, and HE-35A Shionogi, which produce theseantibodies, have been deposited with the Fermentation Research Institute(1-3, Higashi 1 chome, Tsukuba-Shi, Ibaraki-Ken, Japan) since Feb. 27,1990 under the accession Nos. FERM P-11381, FERM P-11382, FERM P-11383and FERM P-11384, respectively, and have been deposited with the sameunder the terms of Budapest Treaty since Apr. 4, 1991 with the accessionNos. FERM BP-3342, FERM BP-3343, FERM BP-3344 and FERM BP-3345,respectively.

The monoclonal antibodies according to the present invention do notreact with any of human IgA, IgG, and IgM, while they react highly withhuman IgE. λ and human IgE.κ, that is, specifically to human IgE.

Monoclonal antibody HE-22A did not react with human IgE which have beentreated with endoglycosidase F capable of cleaving sugar chains ofglycoproteins, thereby being deduced to recognize the structure closelyrelated to sugar chains of human IgE.

Monoclonal antibody HE-35A has significantly low reactivity toheat-modified human IgE and, therefore, recognizes Dε2 domain of humanIgE since the structure of Dε2 domain of human IgE is modified byheating.

Monoclonal antibodies HE-39E and HE-69B recognize Dε1 domain of humanIgE since their binding to human IgE was inhibited by commerciallyavailable monoclonal anti Dε1 antibody (Serotech). However, each one ofmonoclonal antibodies HE-39E and HE-69B does not inhibit the binding ofthe other to human IgE, thus recognizing different regions of Dε1 domainfrom each other.

Thus, any one of the monoclonal antibodies according to the presentinvention binds to human IgE at a region different from regions for theother antibodies without receiving any interference from the bindings ofthe other antibodies to human IgE.

All of the monoclonal antibodies according to the present inventionstrongly bind to intact human IgE but not to human IgE which have beendenatured by guanidine hydrochloride or 2-mercaptoethanol.

All of the monoclonal antibodies according to the present invention areclassified into mouse immunoglobulin G.

Since the monoclonal antibodies according to the present inventionindependently bind to human IgE with different affinities, a mixture ofthe antibodies with controlled reactivity to human IgE can be obtainedby appropriately mixing two or more of the antibodies. For example, insandwich immunoassay using monoclonal antibody HE-69B immobilized, IgEof wide range concentrations can be determined with controlledcalibration curve when a mixture of the monoclonal antibodies HE-22A,HE-35A, and HE-39E (2 to 7:1:5 to 20, as protein concentration), whichhave equal specific activity of label per antibody, was employed as alabelled antibody. Thus, a monoclonal antibody mixture which has adesired reactivity with human IgE can be obtained by mixing at least twodifferent antibodies selected from the group consisting of HE-22A,HE-35A, and HE-39E of the present invention.

Also provided by this invention are the monoclonal antibodies and themixture thereof which have been labelled with radioisotopes such as ¹²⁵I, enzymes such as horse radish peroxidase, or other known labelcompounds such as biotin and avidin.

The present invention further provides hybridoma cell lines producingthe monoclonal antibodies described above.

The present invention also provides a method for an immunoassay of humanIgE comprising:

immobilizing a monoclonal antibody selected from the group consisting ofmonoclonal antibodies HE-22A, HE-35A, HE-39E and HE-69B described above;

contacting the immobilized antibody with a sample containing human IgEto bind the immobilized antibody to the human IgE; and

binding the human IgE which has been bound to the immobilized antibodyto a labelled antibody which is selected from said group and isdifferent from that immobilized.

Any one of the monoclonal antibodies according to the present inventionrecognizes a site different form the others and does not inhibit thebinding of the others to IgE, whereby such a sandwich immunoassay can beeffected. As shown in Examples described below, any one of theantibodies can be used for immobilization and any one of the other threecan be used as a labelled antibody.

Furthermore, the present invention provides an immunoassay of human IgEcomprising:

immobilizing a monoclonal antibody HE-69B described above;

contacting the immobilized antibody with a sample containing human IgEto bind the immobilized antibody to the human IgE; and

binding the labelled mixture of monoclonal antibodies as mentioned aboveto the human IgE which has been bound to the immobilized antibody. Thismethod is preferable since IgE of wide range concentrations can bedetermined with a linear calibration curve.

The present invention still further provides an immunoassay of anantigen-specific human IgE antibody comprising:

contacting an immobilized antigen with a sample containing human IgE;

binding a human IgE antibody specific to the antigen contained in thesample to the immobilized antigen; and

binding a labelled monoclonal antibody to the human IgE antibody whichhas been bound to the immobilized antigen.

For example, when an acarian antigen is used as an immobilized antigen,acarian-antigen specific IgE in a sample such as human blood canexclusively be determined.

A concentration of IgE in the sample assayed can be calculated by usinga calibration curve made by using IgE solutions of fixed differentconcentrations.

EXAMPLE 1

(1) Immunization

Human IgE (200 μg) in Freund's complete adjuvant was administeredintraperitoneally to each of 3 female BALB/c mice of 8 weeks old andafter 3 weeks further 20 μg of human IgE in a form of alum precipitationsuspension was intraperitoneally administered to each mouse forsuccessive 3 days.

(2) Cell fusion

On the next day of the final immunization, spleens were removed from 3mice and a cell suspension thereof was prepared using RPMI medium. TheSpleen cells (3×10⁸) thus obtained and NS-1 myeloma cells (9×10⁷) weremixed and centrifuged to obtain a pellet, to which 1 ml of 50%polyethylene glycol (average molecular weight of 4000) was added slowlywhile stirring, followed by stirring for another 1 minute, 1 ml of RPMImedium was added over 1 minute, and another 1 ml was added, then further7 ml was added over 3 minutes. After centrifugation, the pellet wassuspended in 60 ml of RPMI medium containing 15% fetal bovine serum(complete RPMI medium), and then 0.1 ml of the suspension was placed toeach well of total 6 plates of 96-well microplates, which were incubatedin the presence of 7% CO₂ at 37° C. After 24 hours, 0.1 ml of completeRPMI medium containing 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μMthymidine (HAT medium) was added. On days 2, 3, 5, and 8 afterinitiation of the incubation, 0.1 ml of the supernatant of each culturewas discarded and 0.1 ml of HAT medium was added. On days 11 and 14, 0.1ml of the supernatant was discarded and 0.1 ml of complete RPMI mediumcontaining 100 μM hypoxanthine and 16 μM of thymidine (HT medium) wasadded. In 460 of total 506 wells, hybridoma colonies were developed.

(3) Screening of hybridoma

Hybridomas in the wells were incubated in complete RPMI medium and theproduction of specific antibodies in the supernatant of the culture wasexamined as follows.

A solution (50 μl) of 10 μg/ml human IgE (used in immunization: IgE. λ)dissolved in 0.01M phosphate buffered saline (pH 7.4) was added to eachwell of polystyrene microtiterplates and allowed to stand at 37° C. for1 hour and then 100 μl of 0.01M phosphate buffered saline (pH 7.4)containing 10% fetal bovine serum was added and allowed to stand at 37°C. for 20 minutes for the blocking. The plates were washed with 0.01Mphosphate buffered saline (pH 7.4) containing 0.05% Tween 20 to completehuman IgE-immobilized wells. The human IgE-immobilized wells eachreceived 50 μl of the culture supernatant and were incubated at 37° C.for 1 hour. After washing in the same way as described above, 50 μl ofrabbit anti-mouse IgG (H+L) antibody labelled with horse radishperoxidase was added as a second antibody and incubated at 37° C. for 1hour. After washing as described above, 50 μl of 50 mM citrate buffer(pH 4.5) containing 1.1% hydrogen peroxide and 150 μg/ml azinobis(3-ethylbenzothiazolin-6-sulphonic acid) (ABTS) was added and color wasdeveloped at 25° C. for 5 minutes. The reaction was terminated by adding50 μl of 2 mM sodium azide, and absorbance at 450 nm was determined byusing a spectrophotometer for microplates, and total 145 wells ofhybridomas exhibiting absorbance of 0.5 or higher were selected.

Of the hybridomas producing antibodies against the immunogen human IgE,the reactivity to human IgE (IgE. κ) different from the immunogen and tohuman IgG was examined by ELISA as described above. Then, 40 wells ofhybridomas producing antibodies which reacted strongly with two humanIgEs but not with human IgG were selected. The selected hybridomas werecloned by the limiting dilution method and the supernatant of thehybridoma clones developed was tested for the reactivity of theantibodies to human IgE. λ, human IgE. κ, human IgA, human IgG, humanIgM, and human immunoglobulins λ chain and κ chain by ELISA.

Consequently, 40 clones of the hybridomas which produce monoclonalantibodies specifically reactive to human IgE were obtained.

Among these hybridomas, those which produce monoclonal antibodies havingthe characteristics mentioned above and suitable for the human IgEimmunoassay of the present invention as shown in Experiments describedbelow were selected. Thus, hybridoma HE-39E Shionogi (FERM P-11381),HE-22A Shionogi (FERM P-11382), HE-69B Shionogi (FERM P-11383), andHE-35A Shionogi (FERM P-11384) producing monoclonal antibodies HE-39E,HE-22A, HE-69B and HE-35A, respectively, were obtained.

(4) Preparation of monoclonal antibodies

(a) In vitro incubation

Hybridomas were incubated in complete RPMI medium until growth limit(1×10⁶ cells/ml) and the supernatant of the culture was collected.

(b) In vivo incubation

Hybridoma cells (5×10⁶) were transplanted intraperitoneally to BALB/cmice previously treated intraperitoneally with 0.5 ml of pristane. Afterabout 3 weeks, the mice were observed to ensure the abdominal swellingand then ascitic fluid was collected.

(5) Purification of monoclonal antibodies

Ascitic fluid obtained above was precipitated with 18% sodium sulfatesolution and the precipitation formed was dissolved in 0.01M boratebuffered saline (pH 8.0) and then subjected to dialysis against the samesolution. The monoclonal antibodies (20 mg) obtained by saltprecipitation was dissolved in 2 ml of 0.01M borate buffered saline (pH8.0) and adsorbed to protein A-Sepharose column (Pharmacia AB, 1.5×5cm). The column was eluted with about 50 ml of 0.01M borate bufferedsaline (pH 8.0) to eliminate contaminants, and then with about 100 ml of0.01M citrate buffered saline (pH 4.0) to yield purified monoclonalantibodies.

Experiment 1 Reactivity to human immunoglobulins

Each 50 μl of solutions (10 μg/ml) of two human IgEs, IgE. λ (immunogen)and IgE. κ (Serotech), human IgA (Cappel), human IgM (Miles), human IgG(Miles) and human immunoglobulins L chain type λ and L chain type κdissolved in 0.01M phosphate buffered saline (pH 7.4) was added to eachwell of polystyrene microtiterplates, and allowed to stand at 37° C. for1 hour, and then 100 μl of 0.01M phosphate buffered saline (pH 7.4)containing 10% fetal bovine serum was added and the wells were blockedat 37° C. for 20 minutes. The wells were washed with 0.01M phosphatebuffered saline(pH 7.4) containing 0.05% Tween 20, and wells on whicheach human immunoglobulin was immobilized were obtained. Then, each 50μl of solutions (10 μg/ml) of purified monoclonal antibodies HE-22A,HE-35A, HE-39E, and HE-69B dissolved in 0.01M phosphate buffered saline(pH 7.4) containing 10% fetal bovine serum was added and allowed toreact for 1 hour at 37° C. After washing in the same way as describedabove, color development was conducted as described in Example 1-(3) todetermine the absorbance.

As shown in Table 1, each of monoclonal antibodies HE-22A, HE-35A,HE-39E, and HE-69B reacted with two human IgEs but not with the otherhuman immunoglobulins.

Experiment 2 Iodine (¹²⁵ I)-labelling of monoclonal antibodies

A solution (50 μl) of 40 μg/ml of 1, 3, 4, 6-tetrachloro-3α,6α-diphenylglycoluryl (IODO-GEN, Pierce Chemical) in dichloromethane wasplaced in a glass tube, which was then allowed to stand for dryness.Each 25 μl of 2 mg/ml solution of monoclonal antibody HE-22A, HE-35A,HE-39E, or HE-69B dissolved in 0.1M phosphate buffer solution (pH 7.4)was added to each glass tube and then 0.5 mCi/5 μl radioactive sodiumiodide (Na¹²⁵ I) was added and agitated gently at room temperature for15 minutes. The reaction solution obtained was applied to Sephadex G-25column (Pharmacia, 1.6×18 cm) and eluted with phosphate buffered saline.Earlier fractions were collected to obtain ¹²⁵ I-labelled monoclonalantibodies HE-22A, HE-35A, HE-39E, and HE-69B.

Experiment 3 Horse radish peroxidase (HRP)-labelling of monoclonalantibodies

HRP (5 mg) was dissolved in 2 ml of 0.01M acetate buffer solution and 2mg of sodium methaperiodate dissolved in 0.2 ml of the same buffer wasadded and allowed to react at 25° C. for 20 minutes. The reactionsolution obtained was applied to Sephadex G-25 column (1.6×18 cm) andeluted with 0.001M acetate buffer solution (pH 4.5). According to theabsorbance at 413 nm, initial fractions were collected to yield 1.4 mgof activated HRP, to which 2 mg of HE-22A, HE-35A, HE-39E or HE-69Bdissolved in 1.2 ml of 0.1M carbonate buffered saline was added andallowed to react at 25° C. for 2 hours. An aqueous solution (0.1 ml) of0.2 mg of sodium borohydrite was added and allowed to react at 4° C. for2 hours. The resulting mixture was applied to Sephadex G-25 column(1.6×18 cm), which was eluted with 0.01M phosphate buffered saline (pH7.4). Initial fractions (4 ml) were collected and applied to SephacrylS-200 column (Pharmacia, 1.6×70 cm), which was then eluted with 0.0Mphosphate buffered saline (pH 7.4). Initial fractions were collected toyield each of HRP-labelled monoclonal antibodies HE-22A, HE-35A, HE-39E,and HE-69B.

Experiment 4 Reactivity with heat-treated IgE

A solution (1.5 ml) of 10 μg/ml of human IgE in 0.01M phosphate bufferedsaline (pH 7.4) containing 10% fetal bovine serum was heated at 56° C.for 2 hours.

In the same way as in Experiment 1, prepared were plates having wells onwhich monoclonal antibodies HE-22A, HE-35A, HE-39E, and HE-69B each wasimmobilized. Then, 50 μl of 500, 167, 56, 19 or 2.1 ng/ml solution ofheat-treated human IgE was added to each well and allowed to react at37° C. for 1 hour. As a control, untreated human IgE of the sameconcentrations was used. After washing, 50 μl of 10 μg/ml solution ofHRP-labelled monoclonal antibody ID-15F (reactive with an idiotype partof the antigen human IgE) was added and allowed to react at 37° C. for 1hour. After washing, color development was effected in the same way asin Example 1-(3) to determine the absorbance.

As shown in FIG. 1, monoclonal antibodies HE-22A and HE-69B well reactedwith heat-treated human IgE, while HE-35A exhibited significantlyreduced reactivity with heat-treated human IgE. It is known that suchheat treatment causes the modification of the structure of IgE atDε2region. Thus, HE-35A recognizes the heat-sensitive Dε2 region.

Experiment 5 Reactivity against IgE treated with endoglycosidase F

Endoglycosidase F is an enzyme which cleaves sugar chains ofglycoproteins.

A solution (0.1 ml) of 5 mg/ml of human IgE in physiological saline wasadmixed with70.12 ml of 8.3 U/ml of endoglycosidase F in 0.1M phosphatebuffer solution (pH 6.1) containing 50 mmol/L sodiumethylenediaminetetraacetic acid, 0.05% sodium azide, 0.5% sodiumdodecylsulfate, and 0.05% Tween 20 and allowed to react at 37° C. for 20hours to give endoglycosidase F-treated human IgE.

In the same way as in Example 1-(3), endoglycosidase-treated human IgEwas immobilized on wells of plates. As a control, intact humanIgE-immobilized plates were prepared. Then, 50 μl of a solution ofHRP-labelled monoclonal antibody HE-22A, HE-35A, HE-39E, or HE-69B in0.01M phosphate buffer solution (pH 7.4) containing 10% fetal bovineserum was added to each well and allowed to react at 37° C. for 1 hour.After washing, color development was carried out in the same way as thatused in Example 1-(3) to determine the absorbance.

As shown in Table 2, monoclonal antibodies HE-35A, HE-39E, and HE-69Breacted with endoglycosidase F-treated human IgE, while HE-22A did not.Therefore, HE-22A recognizes the structure closely related with sugarchains of IgE.

Experiment 6 Reactivity of monoclonal antibodies in inhibition test

Human IgE-immobilized plates were prepared in the same way as that inExample 1-(3). Then, 50 μl of 10 μg/ml solution of HRP labelledmonoclonal antibody HE-22A, HE-35A, HE-39E, or HE-69B and 50 μl solutionof unlabelled monoclonal antibodies HE-22A, HE-35A, HE-39E, HE-69B,anti-Dε1 antibody, or anti-Cε4 antibody (2000, 500, 125, 31.2, 7.8, or 0μg/ml) was added to each well and allowed to react with 37° C. for 1hour. After washing, color development was carried out in the same wayas that used in Example 1-(3) to determine the absorbance.

As shown in Table 3, each of HE-22A, HE-35A, HE-39E, and HE-69Binhibited the same antibody on the reaction with human IgE, whereas eachof them did not inhibit the other antibodies on the reaction. HE-39E andHE-69B inhibited monoclonal anti-Dε1 antibody on the reaction. Noantibody was inhibited by monoclonal anti-Cε4 antibody.

Experiment 7 Reactivity with denatured IgE

A solution (0.2 ml) of 5 mg/ml human IgE in physiological saline and anaqueous solution (0.2 ml) containing 6M/L guanidine hydrochloride and 4%2-mercaptoethanol was admixed and allowed to react at 60° C. 2 hours toyield denatured human IgE.

In the same way as that used in Example 1-(3), monoclonal antibodiesHE-22A, HE-35A, HE-39E, and HE-69B each was immobilized on plates. Afterwashing, 50 μl of 1 μg/ml solution of denatured human IgE obtained abovein 0.01M phosphate buffered saline containing 10% fetal bovine serum wasadded to each well and allowed to react at 37° C. for 1 hour. As acontrol, 50 μl of 1 μg/ml solution of intact human IgE was added andallowed to react at 37° C. for 1 hour. After washing, 50 μl of 10 μg/mlsolution of HRP-labelled monoclonal antibody HE-22A, HE-35A, HE-39E, orHE-69B in 0.01M phosphate buffered saline containing 10% fetal bovineserum was added and allowed to react at 37° C. for 1 hour. Afterwashing, color was developed in the same way as in Example 1-(3) todetermine the absorbance.

As shown in Table 4, monoclonal antibodies HE-22A, HE-35A, HE-39E, andHE-69B reacted with intact IgE but not with denatured IgE.

Experiment 8 Sandwich assay of IgE

In the same way as that used in Example 1-(3), monoclonal antibodiesHE-22A, HE-35A, HE-39E, and HE-69B each was immobilized on plates. Afterwashing, each 50 μl of 10,000, 2,000, 400, 80, 16 and 0 IU/ml solutionsof human IgE in 0.01M phosphate buffered saline (pH 7.4) containing 10%fetal bovine serum was added and allowed to react at 37° C. for 1 hour.After washing in the same way as that used in Experiment 5, reactionwith HRP-labelled monoclonal antibody HE-22A, HE-35A, HE-39E, or HE-69Bwas conducted, followed by color development and determination of theabsorbance.

As shown in FIG. 2, among the combinations of any one of the immobilizedmonoclonal antibodies with any one of the HRP-labelled monoclonalantibodies, the combinations of heterogeneous antibodies served as assaysystems of high reactivity with human IgE, while the combinations ofhomogeneous antibody resulted in assay systems of low reactivity.

Experiment 9 Affinity constant to Human IgE

Mite (dermatophagoides pteronisinus; DP)-specific IgE-positive humansera were mixed to obtain 50 ml of serum pool. This pooled serum (200μl) was placed in a test tube(SHIONOGI TUBE™) SHIONOGI TUBE™ is apolystyrene test tube 1 cm×7 cm, with a polyethylene cap and 1 bead ofDP antigen-conjugated beads (MITE-SPECIFIC IgG TEST: SHIONOGI™)MITE-SPECIFIC IgG TEST: SHIONOGI™ is an immunodiagnostic test for mite(D. pteronisinus)-specific IgG was added and allowed to react at 25° C.for 3 hours while shaking. After washing with 0.01M phosphate buffer (pH7.4) containing 0.05% Tween 20, 200 μl of 10, 5, 2.5, 1.25, 0.625,0.313, or 0.156 μCi/ml solution of ¹²⁵ I-labelled monoclonal antibodyHE-22A, HE-35A, HE-39E or HE-69B in 0.01M phosphate buffer (pH 7.4)containing 10% fetal bovine serum was added and allowed to react at 25°C. for 16 hours while shaking. After washing, radioactivity in each tubewas counted by a γ-counter.

As shown in Table 5, HE-35A, HE-69B, HE-22A, and HE-39E exhibited higheraffinity with DP-specific IgE antibodies in this order, and HE-35A,HE-69B, HE-22A, and HE-39E could bind to a larger number of sites(epitope density) in this order.

Experiment 10 Human IgE assay

A solution (200 ml) of 40 μg/ml of monoclonal antibody HE-69B in 0.01Mphosphate buffered saline (pH 7.4) was applied to 100 particles ofpolystyrene beads (Immunochemical) and allowed to stand at 37° C. for 20hours. After discarding the solution, the beads were washed with 0.01Mphosphate buffered saline (pH 7.4) and admixed with 200 ml of 0.01Mphosphate buffer solution (pH 7.4) containing 0.1% bovine serum albumineand allowed to stand at 37° C. for 3 hours. The beads were washed with0.01M phosphate buffered saline (pH 7.4) containing 0.05% Tween 20 toobtain monoclonal antibody HE-69B-conjugated beads.

Solutions of 5,000, 1,000, 200, 40, 8, 1.6 and 0 IU/ml of human IgE in0.01M phosphate buffered saline containing 10% fetal bovine serum wereprepared and 50 μl of each solution was placed in each tube. To eachtube was added 100 μl of 3.7 μCi/ml solution of ¹²⁵ I- labelledmonoclonal antibodies HE-22A, HE-35A or HE-39E or 100 μl of 3.7 μCi/mlsolution of the mixture of HE-22A, HE-35A, and HE-39E. One particle ofmonoclonal antibody HE-69B-conjugated beads was added to each tube andallowed to react at 25° C. for 3 hours. After washing, radioactivity ineach tube was counted by a γ-counter.

As indicated in FIG. 3, HE-35A, HE-22A, and HE-39E gave the sensitivitycurves for human IgE within human IgE concentration ranges of 0.32 to200 IU/ml (625-fold), 1.6 to 1,000 IU/ml (625-fold) and 40 to 5,000IU/ml (125-fold), respectively. On the other hand, the mixture of ¹²⁵I-labelled monoclonal antibodies HE-22A, HE-35A, and HE-39E of certainprotein ratio (3:1:6), all of which have equal specific radioactivity,gave the sensitivity curve covering wide range of concentrations ofhuman IgE from 0.32 to 5000 IU/ml (15,625-fold).

                  TABLE 1                                                         ______________________________________                                        Reactivity of monoclonal antibody with human                                  immunoglobulin in ELISA method                                                Clone No.                                                                             IgE/λ                                                                           IgE/κ                                                                           IgA   IgM  IgG   λ                                                                          κ                         ______________________________________                                        HE-22A  ++       ++      -     -    -     -   -                               HE-35A  ++       ++      -     -    -     -   -                               HE-39E  ++       ++      -     -    -     -   -                               HE-69B  ++       ++      -     -    -     -   -                               ______________________________________                                         ++: Highly positive                                                           +: Positive                                                                   -: Negative                                                              

                  TABLE 2                                                         ______________________________________                                        Reactivity of monoclonal antibody with IgE treated                            with endoglycosidase F                                                        Antigen IgE  HE-22A   HE-35A   HE-39E HE-69B                                  ______________________________________                                        (A) IgE      516      214      776    1031                                    (B) IgE treated with                                                                        12      188      1051    965                                       endoglycosidase F                                                          (B)/(A) %     2        88      135     94                                     ______________________________________                                         Absorbance A490 × 1000                                             

                  TABLE 3                                                         ______________________________________                                        Specificity of reactivity of monoclonal antibody                              in inhibition test                                                            Labelled  Immobilized antibody                                                antibody  HE-22A   HE-35A     HE-39E HE-69B                                   ______________________________________                                        HE-22A    ++       -          -      -                                        HE-35A    -        ++         -      -                                        HE-39E    -        -          ++     -                                        HE-69B    -        -          -      ++                                       ______________________________________                                         ++: Highly positive                                                           +: Positive                                                                   -: Negative                                                              

                                      TABLE 4                                     __________________________________________________________________________    Reactivity of monoclonal antibody with denatured IgE                          in sandwich ELISA system                                                      Immobilized antibody                                                          Labelled                                                                           Intact IgE          Denatured IgE                                        antibody                                                                           HE-22A                                                                             HE-35A                                                                             HE-39E                                                                             HE-69B                                                                             HE-22A                                                                             HE-35A                                                                             HE-39E                                                                             HE-69B                                __________________________________________________________________________    HE-22A                                                                             -    ++   ++   ++   -    -    -    -                                     HE-35A                                                                             ++   +    ++   ++   -    -    -    -                                     HE-39E                                                                             ++   ++   -    ++   -    -    -    -                                     HE-69B                                                                             ++   ++   ++   +    -    -    -    -                                     __________________________________________________________________________     ++: Highly positive                                                           +: Positive                                                                   -: Negative                                                              

                  TABLE 5                                                         ______________________________________                                        Affinity constant of monoclonal antibody for mice                             allergen-specific human IgE antibody                                                      Epitope density                                                                           Affinity constant                                     Anti IgE    (mol/l)     (l/mol)                                               ______________________________________                                        HE-22A      6.4 × 10.sup.-10                                                                    1.3 × 10.sup.8                                  HE-35A      1.5 × 10.sup.-9                                                                     1.6 × 10.sup.9                                  HE-39E      2.6 × 10.sup.-10                                                                    1.2 × 10.sup.8                                  HE-69B      1.3 × 10.sup.-9                                                                     1.2 × 10.sup.9                                  ______________________________________                                    

What we claim is:
 1. A composition comprising a mixture of at leastthree of the monoclonal antibodies selected from the group consisting ofmonoclonal antibodies secreted from the hybridomas deposited as FERM BP-3342, FERM BP-3343, and FERM BP-3345.